Biomass is made up of complex chemicals, and the processes of freeing sugars from the lignocellulosic complexes creates a variety of molecules that prove to be toxic to micro-organisms used for fermentation. Beyond acute toxicity, the fermentation conditions themselves can prove stressful to the organisms negatively affecting ethanol yield. Some of these hydrolysate toxins include amides, weak acids, and aldehydes that have synergistic interactions with other stresses in hydrolysate, including acetate and high osmolarity.
Lignocellulosic plant material is a sustainable and renewable source of biomass for bioenergy and biochemical production. Plant cellulose and hemicellulose harbor significant concentrations of sugars that can be used to produce desired compounds through microbial fermentation. In recent years, several technologies have been developed to hydrolyze plant biomass in order to release monomeric sugars (1, 2). For most types of chemical pretreatment, the resulting hydrolysate contains high sugar concentrations, and thus high osmolarity, and also toxic compounds such as weak acids, furans, and phenolics that are generated as a byproduct of chemical hydrolysis. These hydrolysate toxins (HTs) are known to inhibit microbial growth and fermentation; however, the mechanisms of stress tolerance remain unclear for many of these compounds (3-5). Because removal of these inhibitors from the hydrolysate is expensive (6), a focus is to utilize inhibitor-tolerant microorganisms to produce biofuels and chemicals from plant biomass in an economically viable way.
One approach to this problem is to generate hydrolysate-tolerant microbes by engineering stress tolerance based on the mechanism of toxin action. Most studies elucidating inhibitory mechanisms have focused on individual toxins applied in isolation and have established the effects of such toxins. For example, weak acids such as acetic, formic, and levulinic acids inhibit cell growth and fermentation by mechanisms known as weak acid uncoupling and intracellular anion accumulation (7). Weak acids protonated at low pH can diffuse across the plasma membrane whereupon they dissociate to decrease cytosolic pH (8) and consequently stimulate plasma membrane ATPases that consume ATP to pump protons out of the cell (9, 10). Furans such as 5-hydroxymethyl furfural (HMF) and furfural are also common inhibitors found in hydrolysate, formed by the degradation of xylose and glucose, respectively (7). Furan derivatives are thought to decrease ethanol production by directly inhibiting alcohol dehydrogenase (ADH), pyruvate dehydrogenase (PDH) and aldehyde dehydrogenase (ALDH) enzymes (11). In addition, furfural causes the accumulation of reactive oxygen species that broadly damage membranes, DNA, proteins, and cellular structures (12). Cells respond by reducing furans to less inhibitory compounds at the expense of NAD(P)+ reduction; thus the combined presence of furfural and HMF limits cell division and biofuel production (13, 14). Among other inhibitors, phenolics are the most diverse and the least well understood. These compounds are formed during lignin breakdown, and thus their concentrations and identities mainly depend on the source of plant biomass (4, 15). Phenolic compounds exert considerable inhibitory effects by causing the loss of membrane integrity (16, 17), decreasing cellular ATP (18, 19), causing oxidative damage (17), inhibiting de novo nucleotide biosynthesis (20) and inhibiting translation (21). While the effects of individual toxins are becoming clear in some cases, the compounded effects of multiple toxins in hydrolysate are poorly understood (22, 23). Compounded stress is especially important to consider, since microbes encounter multiple inhibitors at the same time during industrial fermentation of lignocellulosic hydrolysates.
In view of the current state of the biofuel industry, particularly ethanol production based on lignocellulosic feedstocks, it can be appreciated that identifying genes related to enhanced biofuel production is a substantial challenge in the field. Accordingly, a need exists in the field to identify additional genes that influence biofuel production in yeast, and consequently engineer recombinant strains of yeast capable of increased biofuel yields from commonly-available feedstocks, including lignocellulosic plant material.